Wound and ulcer treatment with super-oxidized water

ABSTRACT

Super-oxidized water based on hypochlorous acid, such as is obtained by the electrochemical treatment of a saline solution, may be used in the treatment of leg ulcers or other open wounds. Preferably, the pH of the super-oxidized water is in a range of 4 to 7, and the water has a redox potential of &gt;950 mV. Medicaments based on the super-oxidized water may be in liquid or gel form. The super-oxidized water is able to control the microbial population within the wound and at the same time permit cell proliferation.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a divisional of U.S. Pat. Ser. No. 10/084,518, filedFeb. 25, 2002, now abandoned, which is a continuation of InternationalPatent Application No. PCT/GB00/03264, filed Aug. 23, 2000, which waspublished in the English language on Mar. 1, 2001, under InternationalPublication No. WO 01/13926 A2, and the disclosure of which isincorporated herein by reference.

BACKGROUND OF THE INVENTION

This invention relates to mixtures of oxidants which are referred to inthis specification as “super-oxidized water,” a term which is known inthe art.

Super-oxidized water may be used as a sterilizing, disinfecting andbiocidal solution. One form of super-oxidized water is produced by theapplicant under the trademark STERILOX®. This STERILOX super-oxidizedwater is generated at the point of use, for example in a hospital, bypassing saline solution over coated titanium electrodes separated by asemi-permeable ceramic membrane at a current of about 6 to 9 Amps. Anapparatus having coated titanium electrodes separated by asemi-permeable ceramic membrane is disclosed in the specifications of UKPatent Nos. 2253860 and 2274113. The basic structure of the apparatus isdisclosed in GB2253860 and can be used to produce the STERILOXsuper-oxidized water.

STERILOX super-oxidized water contains a mixture of oxidizing species,predominantly hypochlorous acid (HOCI) and sodium hypochlorite. TheSTERILOX super-oxidized water has a pH of 5-7 and an oxidation reductionpotential (redox) of around 1000 mV. The high redox potential allows forthe quick and efficient destruction of microbes (bacteria, viruses,fungi and spores). Hypochlorous acid and hypochlorite are in equilibriumand the position of the equilibrium is determined solely by the pH.

Applicant has found that the resultant super-oxidized water isnon-hazardous, non-irritating and non-sensitizing to the skin,non-irritating to the eyes, not harmful if swallowed and shows noevidence of mutagenic activity.

It is considered that hypochlorous acid exerts its biocidal effect byattacking the surface and plasma membrane proteins, impairing transportof solutes and the salt balance of bacterial cells (Pieterson et al,Water SA, 22(1): 43-48 (1996)). However, it is believed that HOCl doesnot enter freely into eukaryotic cells, which may explain theselectivity of hypochlorous solutions.

The STERILOX process produces an extremely effective sterilizing, cold,non-toxic solution, which is free from highly toxic chemicals and actsagainst a wide variety of bacteria, fungi, viruses and spores. Thegeneration of STERILOX solutions requires only water, electricity andpure, vacuum-dried crystalline salt. Applicant considers that theSTERILOX super-oxidized water will be suitable for a broad range ofapplications in both medical and non-medical environments, such as thepreservation of poultry and fish and general agricultural andpetrochemical uses, the breaking down of bacterial biofilm, watertreatment and general disinfection in medical and veterinaryapplications. The STERILOX super-oxidized water has been found to beparticularly useful for the disinfection of endoscopes which aresensitive to other cold disinfectants, such as peracetic acid, which arecommonly used.

While glutaraldehyde may be used as a reliable disinfecting agent offlexible fiber-optic endoscopes and other heat-sensitive instruments,although being widely practiced in many hospitals, its use can causeasthma and dermatitis in healthcare staff as a result of exposure toglutaraldehyde fumes, hence a predilection to the use of peracetic acidand the relatively recent move towards the use of STERILOXsuper-oxidized water in such applications.

STERTLOX super-oxidized water has been tested and is the subject of twoscientific papers by Selkon et al, Journal of Hospital Infection, 41:59-70 (1999) and Shetty et al, Journal of Hospital Infection, 41:101-105(1999). In these studies, freshly produced STERILOXsuper-oxidized water was found to be highly active against Mycobacteriumtuberculosis, Mycobacterium avium-intracellulare, Mycobacteriumchelonae, Escherichia coli (including type 0157), Enterococcus faecalis,Pseudomonas aeruginosa, Bacillus subtilis var niger spores,methicillin-resistant Staphylococcus aureus, Candida albicans,poliovirus type 2 and human immunodeficiency virus HIV-1.

There has been a recent upsurge in interest in the use of super-oxidizedwater as a disinfectant, because of its rapid and highly biocidalactivity against a wide range of bacteria. Tanaka et al, Journal ofHospital Infection. 34: 43-49 (1996), report the electrolysis of asaline solution to produce a super-oxidized water with a highly acidicpH of 2.3-2.7, which limits its suitability for many applications,particularly the disinfection of endoscopes. The acidic pH of thesuper-oxidized water produced by the method described by Tanaka et al.also precludes its use in other medical indications.

Having carried out trials in a large number of applications, includingthose mentioned above, Applicant turned its attention to the use ofSTERILOX super-oxidized water as a disinfectant of mammalian tissue, inparticular the treatment of open wounds such as leg ulcers.

An article by Cherry in The Prescriber (May 1996) entitled “GP guide tothe care of patients with leg ulcers” states “leg ulcers are notoriouslydifficult to treat successfully and can seriously reduce the patient'squality of life.” Indeed, according to Cherry, “epidemiological studieshave shown that at any given time there are approximately 100,000patients in the UK that have leg ulceration. In treating these patientsit has been estimated that over £39 million per year alone is spent onmaterials used in their ulcer care.”

There are two types of leg ulcers: arterial and venous. Arterial ulcers,which are much harder to treat, are caused by ischaemia, while venousulcers are caused by blood stasis in the veins.

There are many proposals for the management of ulcers, all of which havevarying degrees of success. Successful ulcer management is very muchdependent on the rigid adherence to a program of treatment incombination with effective disinfection of the wound, which reducesbacterial infection and promotes the regeneration of dermal fibroblastsand keratinocytes in the bed of the ulcer which are essential forhealing of the wound and the growth of new tissue. If the bacterialgrowth is not controlled, the wound cannot heal.

The most useful treatment for venous ulcers is the use of compressionbandages together with elevation of the leg(s). This mimics the pumpingaction of the calf muscles which return the blood back to the body andmaximizes the removal of blood from the leg(s). In conjunction withthis, other treatment strategies include the use of topical treatmentssuch as GRANUFLEX® to aid granulation and skin repair, alginates toclean the wound of debris, dry inert dressings to protect the wound (butwhich do not promote healing), and bacteriostatic or bactericidalointments to reduce the infection. While antibiotics have been used toreduce infection in the past, nowadays this is not a treatment of choicedue to the increased risk of antibiotic resistance.

While potassium permanganate (KMnO₄) is an oxidant which has stood thetest of time in the treatment of leg ulcers, it still nevertheless hasthe disadvantages of irritating and injuring newly grown skin andcausing skin discoloration. Known hypochlorites, such as EUSOL(Edinburgh University Solution of Lime) and Daikin's solution, rely on ahigh concentration of hypochlorite ions for their disinfectantproperties. In fact, these compounds are no longer recommended for usedue to their irritant and painful effects and impairment of cell growthwhich outweigh their therapeutic value, resulting in these preparationsfalling out of use. Attempts have been made to reduce the alkalineeffect of the high hypochlorite ion content of these Solutions, e.g. bythe use of suitable buffers, but have been found to be ineffective insuch circumstances.

All this has militated against the use of preparations includinghypochlorites for the treatment of leg ulcers. However, the success indisinfection and sterilization of endoscopes and the known non-irritanteffects of the STERILOX super-oxidized water, have led the Applicant tore-address the treatment of open wounds such as leg ulcers.

BRIEF SUMMARY OF THE INVENTION

As a first step, in vitro tests were carried out on single layers ofcultured human dermal fibroblast cells and keratinocyte cells toascertain whether or not super-oxidized water had any effect on theviability of the cells. The cells were incubated under sterileconditions in a super-oxidized water based on hypochlorous acid andincluding sodium hypochlorite and other oxidized chlorine species,having a pH range from 4 to 7 and a redox potential of around 1000 mV.

Surprisingly, Applicant found that there is an optimum pH at which cellgrowth is not inhibited despite there being other pH levels within therange at which the viability of the cells is impaired. Indeed, in viewof the findings of Tanaka et al, Applicant expected that the lower pHrange would be more effective.

The next step was crucial, because the applicant then had to ascertainwhether or not the in vitro results could not only be replicated in vivobut also that there would be a sufficient biocidal effect to counteractany bacterial activity which would prejudice the viability of new cells.Accordingly, a clinical trial was carried out on a patient with chronicvenous leg ulcers using freshly produced super-oxidized water based onhypochlorous acid having a pH of 5.4.

The patient did not experience any pain, and in fact commented that thetreatment was comfortable and had a soothing effect. There was apositive effect on the bacterial flora as well as the clinicalappearance of the wounds. No adverse effect was observed on thesurrounding skin which, in a number of patients with long-standingulcers, is often sensitive.

Applicant believes that the effects which have been observed can beexplained by the low concentration of oxidized (free available) chlorinepresent in a super-oxidized water based on hypochlorous acid. This is incontrast to commonly known hypochlorite solutions which owe theirbiocidal activity to a high concentration of free chlorine (includinghypochlorite) as indicated by their characteristic smell.

It could be said that Applicant has discovered a principle, which isthat a balance between the biocidal effect and non-inhibition of cellgrowth enables the microbial population present in a wound to becontrolled such as to allow cell growth to occur.

In order to carry this principle into effect and from one aspect of theinvention, there is provided a super-oxidized water which is based onhypochlorous acid, acts as a biocide and allows cell growth. Expressedin another way, the present invention resides in a super-oxidized waterin which the biocidal effect is due to hypochlorous acid and thenon-inhibitory effect on cell growth is dependent on the level of the pHof the hypochlorous acid solution.

From a still further aspect of the invention, in a super-oxidized waterbased on hypochlorous acid and having a pH of 4 to 7, the solution has apH selected in a range of about 4.3 to 6.2.

In a further aspect of the invention, there is provided, for use in thetreatment of, or medicament for, skin ulcers or other open wounds, asuper-oxidized water or other formulation, such as a gel, which is basedon hypochlorous acid, acts as a biocide and allows cell growth. Thesuper-oxidized water or other preparation based on hypochlorous acid mayhave a pH of 4 to 7, with the pH preferably being selected in a range ofabout 4.0 to 6.5, and more preferably in a range of about 4.0 to 6.2.

In any of the aspects of the invention defined above, the super-oxidizedwater has a biocide rate (D Value) of approximately 1 log unit reductionunit of bacillus subtilis spores in less than 1 minute with a 9:1super-oxidized water: innoculum mix. A biocide rate of about 3.4 secondsmay be achieved and is particularly preferred.

The super-oxidized water based on hypochlorous acid may be obtained bythe electrolysis of a saline solution. Accordingly, from another aspectof the invention there is provided a method of obtaining asuper-oxidized water based on hypochlorous acid, the method comprisingpassing the saline solution through an electrochemical cell havingelectrodes separated by a semi-permeable membrane and operating in suchmanner as to produce a super-oxidized water based on hypochlorous acidwhich acts as a biocide and allows cell growth.

In a preferred method of carrying out the invention, the super-oxidizedwater is obtained and the pH level of the solution is adjusted by abuffering action, which involves the use of an alkaline feed from thecell. By using an alkaline solution in this way, the addition ofconventional buffers and the consequent expense is avoided.

While the pH of the super-oxidized water is preferably adjusted to be ina range of about 4.3 to 6.5, Applicant has found that, in the case ofwounds, such as leg ulcers, it is advantageous if the pH is adjusted toabout 5.4.

From a still further aspect, the invention resides in a method ofproducing a medicament containing super-oxidized water as defined abovefor use in the treatment of leg ulcers or other open wounds.

The invention also comprehends, in a super-oxidized water or otherformulation having a pH of 4 to 7, the selection of a pH in a range ofabout 4.3 to 6.5, and preferably about 5.4, that is used for thetreatment of leg ulcers or other open wounds.

By means of any of the aspects of the invention defined above, thedisinfection of wounds such as leg ulcers is readily achieved withoutirritation and pain, the spread of infection is reduced, and cell growthand regeneration are facilitated, thereby enhancing healing. An addedadvantage is that there is no resistance or tolerance developed todisinfection which would occur with antibiotics.

Furthermore, the super-oxidized water of the invention lends itselfreadily to the treatment being carried out in many ways, although it hasbeen found that a hydrobath in which the leg is immersed is soothing andgenerally pleasant for the patient. However, it should be appreciatedthat preparations other than solutions may be used, for example gels.

From yet another aspect, the invention also resides in a method oftreating a human or animal body having a leg ulcer or other open woundusing any of the super-oxidized water based on hypochlorous acid hereinreferred to above and any of the methods defined hereinabove.

Applicant believes that the present invention, in any or all of itsaspects is a breakthrough and will revolutionize the treatment of openwounds, in particular leg ulcers, in a way which has been found to beimpossible hitherto.

BRIEF DESCRIPTION OF THE SEVERAL VIEWS OF THE DRAWINGS

The foregoing summary, as well as the following detailed description ofthe invention, will be better understood when read in conjunction withthe appended drawings. For the purpose of illustrating the invention,there are shown in the drawings embodiments which are presentlypreferred. It should be understood, however, that the invention is notlimited to the precise arrangements and instrumentalities shown. In thedrawings:

FIG. 1 a is a bar graph illustrating the effect on dermal fibroblastproliferation, measured by absorption assay at 3 and 6 days, of variousconcentrations super-oxidized water; and

FIG. 1 b is a bar graph similar to FIG. 1 a, showing the results of theabsorption assay at 6 days.

DETAILED DESCRIPTION OF THE INVENTION

In order that the invention may be more readily understood, referencewill now be made, by way of example, to the accompanying examples, inwhich:

Example 1 describes an in vitro study, which investigated the effect ofsuper-oxidized water based on hypochlorous acid on the proliferation ofcultured human dermal fibroblast cells.

Example 2 describes an in vitro study, which investigated the cytotoxiceffect of super-oxidized water based on hypochlorous acid on culturedhuman dermal fibroblast cells.

Example 3 describes an in vitro study, which investigated the effect ofsuper-oxidized water based on hypochlorous acid on the proliferation ofcultured human keratinocyte cells.

Example 4 describes a clinical trial of super-oxidized water on apatient with chronic leg ulcers.

EXAMPLE 1

Fibroblasts are flattened, irregular-shaped, connective tissue cellswhich are ubiquitous in fibrous connective tissue. They secretecomponents of the extracellular matrix, including collagen, and play animportant role in tissue regeneration.

Three in vitro trials of super-oxidized water based on hypochlorous acidwere carried out on single-layer cell cultures of human dermalfibroblast (HDF) cells to ascertain whether the super-oxidized wateraffected HDF cell proliferation. A range of dilutions of super-oxidizedwater at different pH levels was investigated.

Method

The super-oxidized water used in the trials was the product of theelectrolysis of an aqueous saline solution passed over a mixture ofproprietary catalysts on titanium electrodes to give a mixture ofoxidizing species, particularly hypochlorous acid (HOCl) at aconcentration of about 144 mg/l to 400 mg/l available free chlorine(Cl). The super-oxidized water was produced as required for each test;the apparatus (supplied by Sterilox Medical Limited, Abingdon, UK) wasoperated to give a final solution redox potential of >950 mV asrecommended by the company. Appropriate dilutions of the super-oxidizedwater were made, and the pH of the final solution was adjusted using aphosphate buffer.

For the proliferation assay, HDF cells were seeded in normal (10%) fetalcalf serum (FCS) and Dulbecco's Modified Eagle Medium (DMEM) at 1.5×10³cells/well. After 24 hours the medium was changed to low (0.4%)FCS/DMEM. After a further 48 hours incubation super-oxidized water atvarying concentrations was added to the cells. The viability of thecells was observed, using a standard absorption assay, 3 and 6 daysafter the application of super-oxidized water.

Results

i) Trial 1: HDF cells were incubated with super-oxidized water in arange of dilutions at a pH of4.3. The dilutions used were: 1, ⅓, 1/7,1/14, 1/28, 1/56, 1/112, 1/224, 1/448, 1/896, 1/1792 and 0.

As shown in the accompanying graphs, on both day 3 (FIG. 1 a) and day 6(FIG. 1 b) super-oxidized water dilutions of 1/28 or less significantlyinhibited HDF proliferation or killed the cells. Slight inhibition ofproliferation was seen at a dilution of 1/56. Dilutions of 1/112 to1/448 showed no effect on proliferation, while the 1/896 dilution showedsome cell proliferation and the dilution of 1/1792 showed significantproliferation of HDF cells.

These results show that high concentrations of super-oxidized watersignificantly inhibit HDF proliferation, probably because of theacidity, and therefore toxicity, of the super-oxidized water. The highlevel of proliferation seen with a concentration of super-oxidized waterat 1/1792 may be attributed to other factors.

ii) Trial 2: Using the same conditions as Trial 1, HDF cells wereincubated with super-oxidized water in a range of dilutions at a pH of6.2. The dilutions used were: 1/20, 1/40, 1/60, 1/80, 1/100, 1/120,1/1000, 1/1500, 1/2000, 1/3000, 1/4000 and 0.

No stimulation of proliferation was seen and indeed, inhibition of HDFgrowth was seen with cells incubated with a dilution of super-oxidizedwater of 1/20. However, the higher dilution of 1/40 showed no celltoxicity.

This trial was repeated with the HDF cells seeded in super-oxidizedwater at dilutions of 1, ¼, ⅛, 1/12, 1/16, 1/20, 1/24, 1/28 and 1/32.After both day 3 and day 6, cell damage or inhibition of proliferationwas seen at dilutions of 1/20 and below. However, dilution of more than1/20 showed no damage or inhibition or proliferation.

In conclusion, while the more alkaline pH appears to be less toxic toHDF cells, proliferation of HDF cells is not seen at this pH.

iii) Trial 3: In this trial, two plates of cells were grown always innormal growth medium (10% FCS/DMEM). One plate of cells was treated asdescribed in Trial 1 but after three days of incubation with thesuper-oxidized water, the growth medium was changed from 0.4% FCS/DMEMto 10% FCS/DMEM in order to observe the recovery of the cells.

HDF cells were incubated at 31° C. with super-oxidized water in a rangeof dilutions at a pH of 5.4. The dilutions used were: 1/10, 1/20, 1/40,1/60, 1/80, 1/100, 1/150, 1/1000, 1/1500, 1/2000, 1/4000 and 0.

On day 3 and day 6 cell proliferation was seen in cells incubated withsuper-oxidized water at a dilution of 1/20 or higher in either 0.4% or10% FCS/DMEM. Some levels of proliferation had reached statisticalsignificance. A dilution of 1/10 super-oxidized water inhibited cellgrowth in HDF cells grown in 0.4% FCS/DMEM but not in 10% FCS/DMEM.

After 3 days incubation in 0.4% FCS/DMEM with or without super-oxidizedwater, the medium was changed to 10% FCS/DMEM. The cells that had beenincubated with super-oxidized water showed the same ability to recoverfrom depression of cell growth seen while growing in 0.4% FCS/DMEM ascontrol cells.

This trial was repeated with the HDF cells seeded in super-oxidizedwater at dilutions of 1/7, 1/10, 1/15, 1/20, 1/40, 1/60, 1/100, 1/500,1/1000, 1/2000, 1/4000 and 0. Again, on both day 3 and day 6, cellproliferation was seen with HDF cells grown in 0.4% FCS/DMEM with thedifference seen being statistically significant at most dilutions. Noinhibition of cell growth was seen, even at the dilution of 1/7super-oxidized water. Stimulation of cell growth was also seen in cellsgrown in 10% FCS/DMEM in the presence of super-oxidized water. However,the levels of proliferation did not reach statistical significance.Where cell growth had been impaired by incubation with super-oxidizedwater, recovery was seen, confirming the observations from the first setof experiments.

In summary, HDF cells incubated with super-oxidized water at pH 5.4showed no inhibition of cell growth, even in the presence of a 1/7dilution of super-oxidized water.

Conclusion

The presence of super-oxidized water at a pH of 5.4 does not inhibit HDFcell growth in vitro.

EXAMPLE 2

Three in vitro trials were carried out to investigate the cytotoxiceffect of super-oxidized water based on hypochlorous acid on HDF cells.

Method

The super-oxidized water based on hypochlorous acid was identical tothat described in Example 1.

HDF cells were seeded in 10% FCS/DMEM at 5×10³ cells/well. Afterincubation at 31° C. for 72 hours, dilutions of super-oxidised waterwere prepared in HBSS and added to the cells. The viability of the cellswas ascertained by a standard absorption assay at time intervals of 15minutes up to one hour from the addition of the super-oxidised water.

Results

i) Trial 1: HDF cells were incubated with super-oxidized water in arange of dilutions at a pH of 4.3. The dilutions used were: 1, ⅓, 1/7,1/14, 1/28, 1/56, 1/112, 1/224, 1/448, 1/896, 1/1792 and 0.

No effect on cell viability was seen in the presence of super-oxidizedwater at dilutions of 1/28 or more at any of the time points. A dilutionof 1/14 induced mild damage to the cells while dilutions of 1/7 and lesskilled the cells.

ii) Trial 2: Using the same conditions as trial 1, HDF cells wereincubated with super-oxidized water in a range of dilutions at a pH of6.2. The dilutions used were: 1, ¼, ⅛, 1/12, 1/16, 1/20, 1/24, 1/28,1/32, 1/36, 1/40 and 0.

No significant effect was seen on the viability of HDF cells in thepresence of super-oxidized water at dilutions of 1/20 or more at anytime point. However, dilution of 1/16 or less induced cell damage.

iii) Trial 3: Using the same conditions as Trials 1 and 2, HDF cellswere incubated with super-oxidized water in a range of dilutions at a pHof 4.0. The dilutions used were: 1, ¼, ⅛, 1/12, 1/16, 1/20, 1/24, 1/28,1/50, 1/100, 1/200 and 0.

Dilutions of super-oxidized water at 1/24 and 1/20 induced slight damageto the cells while dilution of 1/16 or less induced cell death.

Conclusion

The results of these trials support the results shown in Example 1 inthat, while super-oxidized water at pH 4.0 to 4.3 and pH 6.2 inducedamage to cultured HDF cells, greater cytotoxic effects are seen at thelower pH.

EXAMPLE 3

In view of the results described in Examples 1 and 2, two in vitrotrials were carried out to investigate the effect super-oxidized wateron human keratinocyte (HK) cell proliferation. The super-oxidized waterused in these trials was identical to that described in Example 1.Keratinocytes are epidermal skin cells that synthesize keratin and,together with dermal fibroblasts, are essential for skin healing.

Trial 1

HK cells (subcultured, P2, FS, 7 years) were seeded at 8×10³ cells/welland incubated at 31° C. in CLONETICS® (Biowhittaker, US) serum-freemedium with complete supplements, hereinafter referred to askeratinocyte growth medium (KGM), in four 24-well plates. After 24 hoursincubation the medium in plates 1 and 2 was changed to CLONETICS®(Biowhittaker, US) serum-free medium without complete supplements,hereinafter referred to as keratinocyte basal medium (KBM).

After a further 48 hours incubation super-oxidized water diluted in KBMat pH 5.4 was added to plates 1 and 2, and super-oxidized water dilutedin KGM was added to plates 3 and 4. The dilutions of super-oxidizedwater were: 1/10, 1/20, 1/50, 1/100, 1/150, and 0. The pH of the finalsuper-oxidized water solution was adjusted using a phosphate buffer.

After incubation for a further 3 days a standard absorption assay wascarried out on plate 3 to observe the viability and growth of the cells.The absorption assay was carried out on plate 4 after a still furthertwo days. Since the cells incubated in KBM did not grow well, plates 1and 2 were discarded.

The absorption assay showed that, on both day 3 and day 5, cellproliferation had occurred in the presence of all dilutions ofsuper-oxidized water. At day 5, the level of cell proliferation hadreached a significant level compared to cell growth in the absence ofsuper-oxidized water.

Trial 2

In view of the fact that the HK cells did not grow in KBM and showedsignificant proliferation in the presence of super-oxidized water inKGM, it was decided to use KBM with lower concentrations of supplementsas a holding medium, with or without super-oxidized water.

HK cells (thawed, P2, FS, 7 years) were seeded to six 96-well plates at3×10³ cells/well in KGM. After incubation for 24 hours, the medium inplates 1 and 2 was changed to KBM with 20% supplements, and the mediumin plates 3 and 4 was changed to KBM with 50% supplements.

After incubation for a further 24 hours, super-oxidized water diluted inKBM with 20% supplements was added to plates 1 and 2, super-oxidizedwater diluted in KBM with 50% supplements was added to plates 3 and 4,and plates 5 and 6 received super-oxidized water diluted in completeKGM. The dilutions of super-oxidized water were: 1/7, 1/10, 1/15, 1/20,1/40, 1/60, 1/100, 1/500, 1/1000, 1/2000, 1/4000 and 0.

The cells were incubated for a further 3 days in the presence ofsuper-oxidized water, after which time, a standard absorption assay wascarried out on plates 1, 3 and 5 to ascertain the viability of thecells, and the medium in plates 2, 4 and 6 was changed to KGM. Plates 2,4 and 6 were assayed after a further 48 hours of incubation.

Stimulation of cell proliferation on both day 3 and day 5 was seen inall percentages of KGM supplements. However, the level of stimulationwas not significantly different when compared to control cell growth. Nocytotoxicity was seen even at the low dilution of 1/7 super-oxidizedwater.

Conclusion

Dermal keratinocytes cultured in the presence of KGM and super-oxidizedwater showed enhanced cell proliferation, and no cytotoxicity was seenin the presence of super-oxidized water.

EXAMPLE 4

A preliminary clinical evaluation of super-oxidized water based onhypochlorous acid was carried out on one patient with chronic venousulcers on both left and right legs. The aim of the trial was todetermine whether the bacterial status of the ulcers is altered and thebed of the ulcer improved by treatment with super-oxidized water.

Method

The patient's legs were immersed in 40 liters of super-oxidized water ina hydrobath for fifteen minutes before being allowed to dry. Anintermediate assessment without treatment was carried out after oneweek.

A second treatment with super-oxidized water was repeated after twoweeks in which the patient was subjected to three fifteen-minute washesat approximately three-hour intervals. Post-treatment clinicalevaluation was carried out one and several days after the secondtreatment.

Semi-quantitative microbiological analysis of the leg ulcers was carriedout on swabs taken before and fifteen minutes after treatment withsuper-oxidized water.

Results

After the first treatment, the patient reported that the treatment wascomfortable and free from pain. The appearances of the ulcers on bothlegs were markedly improved when assessed five hours after treatment.

As shown in Table 1, quantitative microbiology showed a reduction in thenumber of colony-forming units in the order of 10² in the right legulceration and a reduction in the order of 10⁴ on the left leg.

TABLE 1 semi-quantitative microbiological analysis of leg ulcers beforeand after treatment with super-oxidized water based on hypochlorousacid. Figures quoted indicate the number of colony-forming units (cfu)per ml. Right Leg Left Leg Pre-treatment 1 × 10⁷ 1 × 10⁷ Post-treatment1 × 10⁵ 1 × 10³

The patient was seen one week after the first treatment and was treatedwith conventional therapy, including potassium permanganate. The effectof these on the appearance of the leg ulcers following treatment did notappear to be as striking as that seen with super-oxidized water.

A second treatment with super-oxidized water was repeated a further weeklater, and similar beneficial results were obtained. In between thetreatment periods the ulcers had become sloughy on both legs.Immediately after the first wash, the ulcer bed was whitish due toeffervescence. A cleansing effect was seen after the later two washes,and a marked improvement was seen with the state of the ulcer 18 hoursafter the first wash.

The patient reported no discomfort to the treatment, the solution in thebath was soothing, and the skin felt a bit tight afterwards. The patientcommented that the tightness started to be felt once cold air wasaccessible to the skin.

Referring to Table 2, quantitative microbiology showed a reduction inthe number of colony-forming units in the order of 102 in the right legulceration and a reduction in the order of 10⁴ on the left leg.

TABLE 2 semi-quantitative microbiological analysis of leg ulcers beforeand after treatment with super-oxidized water based on hypochlorousacid. Figures quoted indicate the number of colony-forming units (cfu)per ml. Right leg organisms Left leg organisms (cfu/ml) found (cfu/ml)found pre-treatment 1 1.9 × 10⁸ Coliforms 4.5 × 10⁶ Coliforms Proteusspp Proteus spp skin flora skin flora post-treatment 1 1.2 × 10⁵Coliforms 4.5 × 10⁴ skin flora skin flora β-haemolytic Proteus sppstreptococci Coliforms Proteus ssp pre-treatment 2 3.0 × 10³ Coliforms1.5 × 10⁴ coliforms Proteus spp skin flora skin flora post-treatment 2  1 × 10³ Coliforms <10 no growth Proteus spp skin flora pre-treatment 3<10 no growth 6.3 × 10³ Coliforms β-haemolytic streptococci skin florapost-treatment 3 3.0 × 10² Coliforms 3.0 × 10³ β-haemolytic skin florastreptococci Proteus spp post 24 hours 2.7 × 10² Coliforms 1.5 × 10⁶β-haemolytic Proteus spp streptococci skin flora Coliforms Proteus spp

CONCLUSION

The main objectives of the clinical study were to examine patientcomfort and safety, as well as the efficiency of a treatment ofsuper-oxidized water based on hypochlorous acid.

The use of antiseptics for cleansing wounds is controversial,particularly with reference to the degree of pain associated with thiskind of treatment. This patient did not experience pain and, in fact,commented on a soothing effect. There was a positive effect on thebacterial flora as well as the clinical appearance of the wounds. Therewas no adverse effect on the surrounding skin which, in a number ofpatients with long-standing ulcers, is often sensitive.

While the invention has been described with reference to the examples inrelation to the treatment of leg ulcers, it should be appreciated thatthe invention has considerably wider applicability. For example, theinvention has applicability to burns, to organ transplants in relationto which current practice is to disinfect organs with antibiotics fortwo weeks before they are used in a patient, to disinfection ofvalve-replacements, and to surface wounds, open wounds and plural cavityinfections which are exhibiting drug-resistance.

It will be appreciated by those skilled in the art that changes could bemade to the embodiments described above without departing from the broadinventive concept thereof. It is understood, therefore, that thisinvention is not limited to the particular embodiments disclosed, but itis intended to cover modifications within the spirit and scope of thepresent invention as defined by the appended claims.

1. A method for treating an open wound in a human or animal bodycomprising administering to the open wound an output solution comprisinghypochlorous acid, said output solution: having a pH of 4 to 7, a redoxpotential of >950 mV, and being obtained by electrochemical treatment ofa saline solution; and wherein the output solution is administered in anamount effective to act as a biocide and permit cell proliferation forwound healing.
 2. The method according to claim 1, wherein the openwound is an ulcer, burn, or surface wound.
 3. The method according toclaim 1, wherein the output solution is in a liquid form and is appliedto the wound by bathing.
 4. The method according to claim 3, wherein theoutput solution is applied to the wound by immersion in a hydrobathcontaining the output solution.
 5. The method according to claim 1,wherein the output solution is in a liquid form and is applied to thewound by spraying.
 6. The method according to claim 1, wherein theoutput solution is in a gel form and is topically applied to the wound.7. The method according to claim 1, wherein the output solution has a pHof 4.0 to 6.5.
 8. The method according to claim 7, wherein the outputsolution has a pH of 4.0 to 6.2.
 9. The method according to claim 8,wherein the output solution has a pH of 4.3 to 6.2.
 10. The methodaccording to claim 9, wherein the output solution has a pH of about 5.4.11. The method according to claim 1, wherein the output solution has aredox potential of about 1000 mV.
 12. The method according to claim 1,wherein the output solution, or a preparation derived therefrom, has abiocide rate (D Value) of approximately 1 log reduction unit bacillussubtilis spores in less than 1 minute with a 9:1 output solution:innoculum mix.
 13. The method according to claim 1, wherein the outputsolution is diluted.
 14. The method according to claim 13, wherein theoutput solution is diluted to an extent that it promotes cellproliferation.
 15. The method according to claim 1, wherein the outputsolution has a pH adjusted to a desired level by using an alkalinesolution output from an electrochemical cell in which the salinesolution is treated.
 16. The method according to claim 1, wherein theoutput solution further comprises a phosphate buffer to adjust a pH ofthe solution to a desired level.
 17. The method according to claim 1,wherein the output solution has an available free chlorine concentrationof about 144 mg/l to 400 mg/l.
 18. A method for treating an open woundin a mammal comprising administering to the open wound an outputsolution obtained by electrochemical treatment of a saline solution, theoutput solution comprising hypochlorous acid, having an available freechlorine concentration of about 144 mg/l to 400 mg/l, and having a pH of4 to 7, wherein the output solution is administered in an amounteffective to act as a biocide and permit cell proliferation for woundhealing.
 19. The method according to claim 18, wherein the open wound isan ulcer, burn, or surface wound.
 20. The method according to claim 18,wherein the output solution is in a liquid form and is applied to thewound by bathing.
 21. The method according to claim 18, wherein theoutput solution is applied to the wound by immersion in a hydrobathcontaining the output solution.
 22. The method according to claim 18,wherein the output solution is in a liquid form and is applied to thewound by spraying.
 23. The method according to claim 18, wherein theoutput solution is in a gel form and is topically applied to the wound.24. The method according to claim 18, wherein the output solution has apH of 4.0 to 6.5.
 25. The method according to claim 18, wherein theoutput solution has a pH of 4.0 to 6.2.
 26. The method according toclaim 18, wherein the output solution has a pH of 4.3 to 6.2.
 27. Themethod according to claim 18, wherein the output solution has a pH ofabout 5.4.
 28. The method according to claim 18, wherein the outputsolution has a redox potential of >950 mV.
 29. The method according toclaim 18, wherein the output solution has a redox potential of about1000 mV.
 30. The method according to claim 18, wherein the outputsolution, or a preparation derived therefrom, has a biocide rate (DValue) of approximately 1 log reduction unit bacillus subtilis spores inless than 1 minute with a 9:1 output solution: innoculum mix.
 31. Themethod according to claim 18, wherein the output solution is diluted.32. The method according to claim 18, wherein the output solution isdiluted to an extent that it promotes cell proliferation.
 33. The methodaccording to claim 18, wherein the output solution has a pH adjusted toa desired level by using an alkaline solution output from anelectrochemical cell in which the saline solution is treated.
 34. Themethod according to claim 18, wherein the output solution furthercomprises a phosphate buffer to adjust a pH of the solution to a desiredlevel.